Extraction and Purification of DNA‐Dependent RNA Polymerase from Rat Liver Nuclei

The enzyme RNA polymerase is difficult to extract from most mammalian cells due to its firm association with chromatin. By a combination of intensive ultrasonic treatment and extraction of the nuclei with 0.75 M NaCl at 0° and pH 8.4 for 1.5 hours approximately 80% of the enzyme can be extracted in a soluble, DNA‐dependent form. The extracted enzyme has been purified by DEAE‐cellulose chromatography and centrifugation on sucrose gradients (5–20%, w/v) containing 10% glycerol. A purification of approximately 250‐fold has been achieved in the peak fraction of the gradient. The enzyme requires all four nucleoside triphosphates, mercaptoethanol, a DNA template and either Mg++ or Mn++, the latter being the preferred ion. Enzyme activity can be stimulated by (NH4)2SO4. No direct stimulation of enzyme activity by cortisol phosphate could be observed.